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Arabinosylation Plays a Crucial Role in Extensin Cross-linking In Vitro.

Chen Y, Dong W, Tan L, Held MA, Kieliszewski MJ - Biochem Insights (2015)

Bottom Line: Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking.Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate.Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, OH, USA.

ABSTRACT
Extensins (EXTs) are hydroxyproline-rich glycoproteins (HRGPs) that are structural components of the plant primary cell wall. They are basic proteins and are highly glycosylated with carbohydrate accounting for >50% of their dry weight. Carbohydrate occurs as monogalactosyl serine and arabinosyl hydroxyproline, with arabinosides ranging in size from ~1 to 4 or 5 residues. Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking. Here, the involvement of arabinosylation in EXT cross-linking was investigated by assaying the initial cross-linking rate and degree of cross-linking of partially or fully de-arabinosylated EXTs using an in vitro cross-linking assay followed by gel permeation chromatography. Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate. Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.

No MeSH data available.


Hyp-Ara profiling of partially deglycosylated EXTs. (A) Top to bottom: RSH, RSHAra, and RSHpH2; (B) top to bottom: TOMP1, P1Ara, and P1pH2. Each Hyp-arabinosides were present in two peaks due to two configurations of Hyp (cis or trans) generated during base hydrolysis.50 The Hyp-Ara4 peaks disappeared after α-arabinofuranosidase treatment, while some Hyp-Ara4 glycans were still present after pH 2.0 HCl deglycosylation. See Table 2 for Hyp-oligoarabinan content quantification in each sample.
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f1-bci-suppl.2-2015-001: Hyp-Ara profiling of partially deglycosylated EXTs. (A) Top to bottom: RSH, RSHAra, and RSHpH2; (B) top to bottom: TOMP1, P1Ara, and P1pH2. Each Hyp-arabinosides were present in two peaks due to two configurations of Hyp (cis or trans) generated during base hydrolysis.50 The Hyp-Ara4 peaks disappeared after α-arabinofuranosidase treatment, while some Hyp-Ara4 glycans were still present after pH 2.0 HCl deglycosylation. See Table 2 for Hyp-oligoarabinan content quantification in each sample.

Mentions: Hyp-Ara profiles of EXTs treated with arabinofuranosidase and pH 2.0 HCl (native RSH and TOMP1 as controls, Fig. 1 and Table 2) were determined, and the results revealed that no Hyp-tetraarabinosides were observed after treatment with arabinofuranosidases, while some Hyp-Ara4 remained after pH 2.0 treatments (Fig. 1).


Arabinosylation Plays a Crucial Role in Extensin Cross-linking In Vitro.

Chen Y, Dong W, Tan L, Held MA, Kieliszewski MJ - Biochem Insights (2015)

Hyp-Ara profiling of partially deglycosylated EXTs. (A) Top to bottom: RSH, RSHAra, and RSHpH2; (B) top to bottom: TOMP1, P1Ara, and P1pH2. Each Hyp-arabinosides were present in two peaks due to two configurations of Hyp (cis or trans) generated during base hydrolysis.50 The Hyp-Ara4 peaks disappeared after α-arabinofuranosidase treatment, while some Hyp-Ara4 glycans were still present after pH 2.0 HCl deglycosylation. See Table 2 for Hyp-oligoarabinan content quantification in each sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4629521&req=5

f1-bci-suppl.2-2015-001: Hyp-Ara profiling of partially deglycosylated EXTs. (A) Top to bottom: RSH, RSHAra, and RSHpH2; (B) top to bottom: TOMP1, P1Ara, and P1pH2. Each Hyp-arabinosides were present in two peaks due to two configurations of Hyp (cis or trans) generated during base hydrolysis.50 The Hyp-Ara4 peaks disappeared after α-arabinofuranosidase treatment, while some Hyp-Ara4 glycans were still present after pH 2.0 HCl deglycosylation. See Table 2 for Hyp-oligoarabinan content quantification in each sample.
Mentions: Hyp-Ara profiles of EXTs treated with arabinofuranosidase and pH 2.0 HCl (native RSH and TOMP1 as controls, Fig. 1 and Table 2) were determined, and the results revealed that no Hyp-tetraarabinosides were observed after treatment with arabinofuranosidases, while some Hyp-Ara4 remained after pH 2.0 treatments (Fig. 1).

Bottom Line: Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking.Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate.Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, OH, USA.

ABSTRACT
Extensins (EXTs) are hydroxyproline-rich glycoproteins (HRGPs) that are structural components of the plant primary cell wall. They are basic proteins and are highly glycosylated with carbohydrate accounting for >50% of their dry weight. Carbohydrate occurs as monogalactosyl serine and arabinosyl hydroxyproline, with arabinosides ranging in size from ~1 to 4 or 5 residues. Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking. Here, the involvement of arabinosylation in EXT cross-linking was investigated by assaying the initial cross-linking rate and degree of cross-linking of partially or fully de-arabinosylated EXTs using an in vitro cross-linking assay followed by gel permeation chromatography. Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate. Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation.

No MeSH data available.